HIV-1 Tat activates viral transcription and limited Tat-transactivation correlates with latency establishment. We postulated a ?block-and-lock? functional cure approach based on properties of the Tat-inhibitor didehydro-Cortistatin A (dCA). HIV-1 transcriptional inhibitors could block ongoing viremia during antiretroviral therapy (ART), locking the HIV promoter in persistent latency. We investigated this hypothesis in human CD4+T cells isolated from aviremic individuals. Combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation. We show that dCA mediates epigenetic silencing by increased nucleosomal occupancy at Nucleosome-1, restricting RNAPII recruitment to the HIV-1 promoter. The efficacy of dCA was studied in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence. Adding dCA to ART suppressed mice systemically reduces viral mRNA in tissues. Moreover, dCA significantly delays and reduces viral rebound levels upon treatment interruption. Altogether this work demonstrates the potential of ?block-and-lock? cure strategies. A Tat inhibitor is unlike any other HIV inhibitor, as duration of treatment impacts the outcome, because of the feedback nature of the Tat-TAR activity and because epigenetic marks deposited at the HIV-1 promoter accrue over time. We hypothesized that over time transcriptional repression could be pushed past a certain threshold where viral reactivation from latency is extremely difficult to overcome, blocking-and-locking HIV into sustained latency. The additive activity of dCA also supports the notion that adding Tat inhibitors to front-line treatment might lead to faster suppression and potentially reduce the size of the established reservoir. It is emerging from studies of individuals on very early ART treatment that a smaller reservoir size directly translates into better viral control (14). The genetic barrier to viral resistance to dCA in vitro was investigated, and unexpectedly but not too surprising, mutations in Tat and TAR were not found, since these are extremely conserved. Instead, viruses resistant to dCA developed very high Tat-independent basal transcription. We identified a combination of mutation in the LTR promoter that increased basal transcriptional activity, and modifications in Nef and Vpr that increased NF-?B activity. We hypothesize these viruses may not develop in vivo, we reason their increased transcription fitness and inability to control entry into latency may ultimately be detrimental, leading to high cytopathic effects and/or clearance by the immune system. Looking ahead we have three main goals: 1) using BLT mice to understand the relationship between dCA treatment time and reductions in residual viral RNA production and how that translates in delaying viral rebound after treatment interruption; 2) study the impact of dCA as front-line therapy on the size of the established viral reservoir during acute phase treatment; and 3) study mechanisms of viral resistance to dCA in vivo.